Advances in molecular biology have enhanced the ability of scientists to manipulate the genome of animals and plants. Genes controlling various aspects of plant and animal molecular processes may be identified and isolated from the genomes of those respective organisms. For example, genes conferring antibiotic, herbicide, and insect or drought resistance have been isolated from various organisms. Even more important is the ability to take a gene isolated from one organism and introduce said gene into another organism (heterologous transformation). This integration may be accomplished even where the recipient organism is from a different phylum, genus or species from that which the gene was derived.
Generally, plant transformation relies on two approaches for delivery and expression of foreign genes in plants: stable genetic transformation and transient expression. A number of genetic engineering techniques have been employed to stably introduce desired traits into plant genomes. The introduction of these desired traits have been accomplished by means including Agrobacterium infection (Nester et al., 1984), polyethylene glycol (PEG)-mediated DNA uptake (Lorz et al., 1985), electroporation of protoplasts (Fromm et al., 1986) and microprojectile bombardment (Klein et al., 1987). Many plant species may now be transformed stably on a routine basis by using the aforementioned methods or variants thereof (for review see: Christou et al., 1996, Trends Plant Sci. 1, 423-431). Plant transient expression can be accomplished through agro-infiltration, particle bombardment or viral infection (for review see: Fischer et al., 1999, Biotechnol. Appl. Biochem., 30, 113-116).
Transient expression of nucleic acids has great potential as a means to predict how any gene, promoter, expression cassette, or other elements might perform in a stable transgenic plant. The development of a transient expression in planta assay to permit rapid evaluation of heterologous gene expression in plants is highly desirable. The conventional establishment and characterization of a stable transgenic plant line involves a long process often taking more than two years. It would be ideal to have a rapid transient assay method to quickly evaluate how an expression cassette and or its associated elements (i.e. promoter, gene, enhancers) will perform in stable plant lines. For instance, it would be ideal to have a transient method where one could quickly correlate by using transient data the best expression method (i.e. cellular targeting, enhancer combinations, promoter selection, etc) to be employed in stable plant lines. This method could also be utilized to quickly identify expression problems such as protein cleavage, tissue toxicity, unfavorable phenotypes as well as other problems that could be identified prior to investing time and resources to express gene candidates in stable plant lines. Transient expression can be achieved by agro-infiltrating plant tissue with a standard expression cassette under control of a constitutive promoter such as the 35S promoter to drive expression of the gene of interest (Vaquero et al., 1999, Proc. Natl. Acad. Sci. US, 96, 11128-11133). One disadvantage of transiently expressing genes of interest using the agro-infiltration method is that the method results in very low protein expression levels. Low protein expression makes it difficult to correlate how a gene or expression cassette might perform stably in planta. It has been found that the inclusion of post-transcriptional gene silencing suppressors in agro-infiltration, such as p19 or HcPro, results in a 50 fold increase in transient expressed protein (Voinnet et al., 2003, Plant J., 33, 549-556). Though the transient protein expression levels are higher, agro-infiltrated transient expression employing post-transcriptional gene silencing suppressors can in some cases be inconsistent in regards to protein expression and not at all predictive of how a given gene or expression cassette might perform in stable plants. Viral vectors may also be used to transiently express proteins of interest. Viral vectors overcome the problem of producing high transient expression levels (for review see: Porta & Lomonossoff, 1996, Mol. Biotechnol., 5, 209-221; Yusibov et al., 1999, Curr. Top. Microbiol. Immunol., 240 81-94). However, the use of viral vectors to transiently express a protein in plants is limited by a narrow host range in terms of their best performance as well as limitations to gene size. There is also the issue that no transient assay method has been identified to work consistently across a variety of plant species. Monocots are an especially difficult group of plants to consistently express genes of interest transiently in a manner that transient data may be used as a predictive indicator of how a gene or expression cassette may perform in stable plant lines. Of particular interest, would be a transient expression in planta assay method that could work in cereal crops (e.g. maize or wheat), sugarcane, sugar beet, soybean, rice as well as other commercially important crops.